research article an upgrade on the rabbit model of...

Research ArticleAn Upgrade on the Rabbit Model of Anthracycline-InducedCardiomyopathy: Shorter Protocol, Reduced Mortality, andHigher Incidence of Overt Dilated Cardiomyopathy

Jesús Talavera,1 Alejandro Giraldo,2 María Josefa Fernández-Del-Palacio,1

Obdulio García-Nicolás,3,4 Juan Seva,3 Gavin Brooks,2 and Jose M. Moraleda5

1Departamento de Medicina y Cirugıa Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de ExcelenciaInternacional Regional “Campus Mare Nostrum”, 30100 Murcia, Spain2School of Biological Sciences, Institute for Cardiovascular and Metabolic Research, University of Reading, Reading RG6 6AS, UK3Departamento de Anatomıa y Anatomıa Comparada, Facultad de Veterinaria, Universidad de Murcia,Campus de Excelencia Internacional Regional “Campus Mare Nostrum”, 30100 Murcia, Spain4Institute of Virology and Immunology (IVI), Sensemattstrasse 293, 3147 Mittelhausern, Switzerland5Unidad de Trasplante Hematopoyetico y Terapia Celular, Departamento de Hematologıa,Hospital Universitario Virgen de la Arrixaca, IMIB, Universidad de Murcia, 30120 Murcia, Spain

Correspondence should be addressed to Jesus Talavera; [email protected] and Alejandro Giraldo; [email protected]

Received 21 August 2015; Revised 24 November 2015; Accepted 26 November 2015

Academic Editor: Peter M. Becher

Copyright © 2015 Jesus Talavera et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Current protocols of anthracycline-induced cardiomyopathy in rabbits present with high premature mortality and nephrotoxicity,thus rendering them unsuitable for studies requiring long-term functional evaluation of myocardial function (e.g., stem celltherapy). We compared two previously described protocols to an in-house developed protocol in three groups: Group DOX2received doxorubicin 2mg/kg/week (8 weeks); Group DAU3 received daunorubicin 3mg/kg/week (10 weeks); and Group DAU4received daunorubicin 4mg/kg/week (6 weeks). A cohort of rabbits received saline (control). Results of blood tests, cardiactroponin I, echocardiography, and histopathology were analysed. Whilst DOX2 and DAU3 rabbits showed high prematuremortality (50% and 33%, resp.), DAU4 rabbits showed 7.6% premature mortality. None of DOX2 rabbits developed overt dilatedcardiomyopathy; 66% of DAU3 rabbits developed overt dilated cardiomyopathy and quickly progressed to severe congestive heartfailure. Interestingly, 92% of DAU4 rabbits showed overt dilated cardiomyopathy and 67% developed congestive heart failureexhibiting stable disease. DOX2 andDAU3 rabbits showed alterations of renal function, withDAU3 also exhibiting hepatic functioncompromise. Thus, a shortened protocol of anthracycline-induced cardiomyopathy as in DAU4 group results in high incidence ofovert dilated cardiomyopathy, which insidiously progressed to congestive heart failure, associated to reduced systemic compromiseand very low premature mortality.

1. Introduction

Anthracyclines (AC) such as doxorubicin and daunorubicinare regarded as one of the most effective chemotherapeuticgroups ever developed for the treatment of malignancies,with a broad spectrum of action encompassing solid andhaematologic tumours [1, 2]. Unfortunately, AC-inducedcardiomyopathy (AICM) is a frequent toxic consequence ofAC. It is estimated that in the USA alone there are at least

10 million cancer survivors, with a similar number in Europe[3, 4]. Thus, the recent success of chemotherapy in clinicaloncology means that the population affected by AICM willlikely increase substantially in the future.

Several animal models and protocols for the inductionof AICM in rodents (e.g., mice and rats) and lagomorphs(e.g., rabbits) have been published over the past decades. Ofnote, rabbit models of cardiac disease appear to have severaladvantages over animalmodels of other species. For example,

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 465342, 13 pageshttp://dx.doi.org/10.1155/2015/465342

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whilst being medium size animals, rabbits maintain a cellularelectrophysiology and Ca+2 transport system, much like inthe human or larger animals (e.g., dogs and pigs), which isnot the case for mice and rats [5]. One of the first long-term animal models of AICM was established in rabbits byadministering daunorubicin and demonstrating myocardialdamage and fibrosis [6]. Subsequent studies in rabbits char-acterized the cumulative and delayed nature of myocardiallesions secondary to administration of doxorubicin 2mg/kgweekly at different time points [7]. Most studies using rabbitmodels of AICM have focused on the evaluation of potentialcardioprotective agents aimed at preventing AICM develop-ment [8–13]. However, with the current trend of increasedlife expectancy of cancer patients and their associated risk oflong-term cardiovascular sequelae, the preclinical assessmentof novel therapies (e.g., stem cell therapy) to treat thiscondition requires the refinement of current animal modelsto maximise their potential.

One of the main disadvantages of current protocols ofinduction ofAICM in rabbits is the highmortality rate duringthe induction period (30–70%) [10, 14–16]. This not onlyincreases the number of animals required but also limitstheir utility in the evaluation of the beneficial effects ofnovel therapies for AICM. Another disadvantage is that eventhough cardiac toxicity is readily reproducible with someprotocols using daunorubicin or doxorubicin concomitantsystemic toxicity (e.g., nephrotoxicity) occurs with theseprotocols of induction [10, 17–20]. This systemic toxicity isoften responsible for the premature death of the animalseven before they can develop clinically evident signs ofovert dilated cardiomyopathy (DCM) and congestive heartfailure (CHF). The availability of an experimental modelthat provides animals in a stable clinical stage of heartfailure would offer a valuable tool to researchers interestedin evaluating the benefits of therapies that require long-termfollow-up of the animals.

Our goal has been to study the benefits of stem celltherapy in AICM, but in the process we have come across thepitfalls of current protocols of induction, thus motivating usto develop our own in-house protocol. Since cardiac toxicityis directly related to the cumulative dose administered tothe subject of study [21] and rabbits are very sensitive tomyocardial damage by AC [7], we hypothesised that increas-ing the weekly dose of daunorubicin and reducing the lengthof the protocol of induction could result in a rabbit modelof AICMpotentially exhibiting reduced nonspecific toxicitiesand lower mortality. Of the several experimental protocolstrialled, here we report the incidence of overt DCM, CHF,premature mortality, and associated systemic toxicities whenusing an in-house developed shortened protocol (daunoru-bicin 4mg/kg per week for up to 6 weeks) compared to otherprotocols reported in the literature which are frequently usedfor the evaluation of cardioprotective agents.

2. Materials and Methods

2.1. Animals. The experiments in the present study wereperformed in accordance with Directive 2010/63/EU of theEuropean Commission and were approved by the Ethical

Research Committee of the University of Murcia, Spain.A total of 37 New Zealand Rabbits (2 months old, 1.5–2.0 kg weight with 1 : 1 ratio of males/females) were randomlyallocated into one of the following three groups: 6 rabbitswere injected I.V. 0.9% saline weekly for up to 10 weeksand constituted the age matched controls (control group);12 rabbits constituted DOX2 group receiving doxorubicin(Tedec-Meiji Farma, Madrid, Spain) 2mg/kg in weekly intra-venous injections for 8 weeks; 6 rabbits constituted the DAU3groupwhichwas injectedwith daunorubicin (Daunoblastina,Pfizer, Madrid, Spain) 3mg/kg in weekly for 10 weeks; and 13rabbits constituted the DAU4 group (daunorubicin 4mg/kgper week for 6 weeks). Due to inherent differences in theinduction times for each protocol, three time points weredefined for the comparisons of biochemical, haematological,and echocardiographic analyses between study groups: thefirst time point is the baseline time point, just before startingthe first administration of anthracycline in treated groupsor saline in control group; the second time point is theintermediate time point, two weeks before the end of theprotocol; thus intermediate time points were at 8 weeks forcontrol group, 6 weeks for DOX2 group, 8 weeks for DAU3group, and 4 weeks for DAU4 group; and the third timepoint is the final time point, the last measurement taken uponcompletion of the protocol.

2.2. Mortality. The premature death of the animals dur-ing the period of induction of cardiomyopathy (i.e., deathbefore completing the corresponding experimental inductionprotocol of weekly AC injections) was classified as follows:(1) deaths due to systemic toxicity (i.e., directly related tothe administration of AC) which manifested as diarrhoea,weight loss, and emaciation and included animals that diedor required euthanasia for this cause, (2) deaths secondary toCHF (see below), which were confirmed either by echocar-diogram and/or by autopsy, (3) other causes such as respi-ratory failure during anaesthesia or animals that requiredeuthanasia (e.g., spontaneous spinal fracture). Kaplan-Meiersurvival analysis was performed throughout the inductionperiod and up to two weeks after completion of the respectiveinduction protocol for each group.

2.3. Incidence of Overt DCM and CHF. For the analysisof incidence of overt DCM and development of CHF inthe different groups of study, these were defined as follows.Overt DCM is defined as unequivocal echocardiographicsigns of cardiac remodelling and/or functional impairmentobjectively assessed by the presence of several of the followingfindings: eccentric hypertrophy of cardiac chambers, loss ofthe oval shape of the left ventricle, ventricular wall thinning,increased left atrial-to-aortic root ratio> 1.5 (2D-mode, shortaxis view), fractional shortening (FS) < 20%, left ventricularejection fraction (LVEF) < 40%, and presence of atrioventric-ular valve regurgitation (assessed by colour and/or spectralDoppler) (Figures 1(c) and 1(d)). On the other hand, CHFwas defined as echocardiographic and/or postmortem evi-dence of pleural, pericardial, and peritoneal effusions and/orpulmonary oedema in conjunction with echocardiographic

BioMed Research International 3

(a) (b)

(c) (d)

(e) (f)

Figure 1: Echocardiograms in 2D and M-mode during induction of AICM. Echocardiograms in 2D parasternal long axis view (a, c, e) andM-mode of the left ventricle (LV) at the level of the papillary muscles (b, d, f). (a-b) Baseline, normal rabbit. (c-d) Rabbit with overt DCM.(e-f) Rabbit with CHF showing 4-chamber dilatation, pericardial effusion (PE), and severely reduced LV contractility. RV, right ventricle; LA,left atrium; RA, right atrium.

signs of overt DCM and/or severe cardiomegaly confirmedby postmortem study (Figures 1(e) and 1(f)).

2.4. Administration of Drugs and Blood Sampling. Animalswere immobilised in acrylic restrainers to reduce stressand facilitate the administration of drugs. AC were dilutedaccording to the weight of the animal in 8mL of 0.9% sterilesaline for I.V. injections in AC treated groups, whilst incontrol group 8mL of 0.9% sterile saline without AC wereinjected at each time point. After hair clipping of the earand careful asepsis, a 24G catheter was advanced into one

of the marginal auricular veins and fixed with micropore tothe skin. A winged needle (21 G) with prolongator was theninserted into the catheter and AC were administered at arate of 0.5mL/min. Blood samples from jugular vein werecollected before first dose of AC and then every 2 weeks untilthe final administration at 8 weeks (DOX2 group), 10 weeks(DAU3 group), or 6 weeks (DAU4 group).

2.5. Biochemical and Haematological Study. Haematologicalparameters in plasma samples were analysed using the Advia120 Hematology System (Siemens, Erlangen, Germany).


Biochemical parameters were evaluated in plasma using theanalyser AU2700 (Olympus Corporation, Tokyo, Japan).

2.6. Echocardiographic Study. A transthoracic echocardio-graphic examination using a HD7 XE System, equipped witha 4–12MHz transducer (Philips, Andover, Massachusetts,USA) under light anaesthesia (ketamine (Imalgene, Merial,Villeurbanne, France) 10mg/kg, combined with dexmedeto-midine (Domtor, Esteve, Madrid, Spain) 200𝜇g/kg), wasperformed longitudinally at baseline and every two weeksuntil the end of the study. The procedure was performedby or under direct supervision of a European board-certified veterinary cardiologist (MJFP), in a blinded fashion,according to the recommendations of the EchocardiographyCommittee of the American College of Veterinary InternalMedicine and theAmerican Society of Echocardiography [22,23]. Simultaneous 1-lead electrocardiographic tracings wererecorded during the study. Total circumferential shorteningarea (CSA) was obtained using the following formula: CSA =CSAd − CSAs/CSAd × 100. Fractional shortening (FS (%))was calculated according to the following formula: FS =(LVDd − LVDs)/(LVDd × 100). Left ventricular systolic anddiastolic volumes (LVVd and LVVs) were calculated using theTeichholz formula (7 × (LVD)3)/(2.4 + LVD) and LVEF (%)was calculated according to the following formula: LVEF =(LVVd − LVVs)/(LVVd × 100).

2.7. Cardiac Troponin I Evaluation. Cardiac troponin I (cTnI)levels were determined in plasma samples obtained fromthe jugular vein using the Immulite Kit (Siemens, Germany)according to the manufacturer’s instructions with a detectionlimit <0.049 ng/mL.

2.8. Histopathological Study. Heart tissue blocks were sec-tioned to cover 5 different anatomical regions in ascendingorder: the apex, free wall bellow the papillary muscles,at the level of the papillary muscles, end of papillarymuscles and beginning of chordae tendineae, and at theatrial level [7]. Tissue was fixed for 24 h in 10% formalin,dehydrated with increasing ethanol concentrations, whichwas then substituted for xylene, and finally embedded inparaffin using the Leica TP1050 cyclic tissue processor (LeicaBiosystems GmbH, Germany) and the Tissue-Tek thermalconsole (Sakura, USA). Sections of 5 𝜇m were then obtainedwith a microtome RM2155 (Leica Biosystems GmbH, Ger-many) and stained with haematoxylin-eosin and Masson’strichrome. Two pathologists blinded to study group per-formed the histopathological evaluations. Myocardial lesionswere assigned a score from 1–4 by lesion grade, and theextension of myocardial lesions was classified 1–4 as pre-viously described [7]. A similar lesion grade scoring (1–4)and classification of extension of lesions (1–4) system wasfollowed for kidney and liver tissue.

2.9. Statistical Analysis. Statistical analysis was performedusing SPSS Statistics version 19 for Windows and GraphPadPrism 6. Data are expressed as means ± SEM. One-wayANOVA with Tukey’s post hoc test or paired 𝑡 test/Wilcoxon

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Figure 2: Kaplan-Meier survival curves.

signed-rank test or two-way ANOVA to assess the globaleffect of sex and treatment, where appropriate, was per-formed. Survival curves were plotted using Kaplan-Meieranalysis (Log-Rank withMantel-Cox test). Values of 𝑝 ≤ 0.05were considered statistically significant.

3. Results

3.1. Mortality. Survival analysis curves are shown in Fig-ure 2. In the DOX2 group, 6 out of 12 animals (50%) diedprematurely (premature mortality defined as death of theanimal during the course of weekly I.V. administrations oneach group) during weeks 3–7 of the induction period (upto 8 weeks), all secondary to systemic toxicity. In the DAU3group, 33.3% died prematurely during the induction period(up to 10 weeks); one of these deaths occurred secondary tosystemic toxicity at 4weeks and the other occurred secondaryto CHF at week 9. In the DAU4 group, only one animalrequired euthanasia at week 5 due to spontaneous spinalfracture resulting in a premature mortality of 7.6% duringthe induction period (up to 6 weeks). No deaths occurred inthe control group. Gender of the animal did not influencemortality in all AC treated groups since this did not differsubstantially from that of whole cohort within each group.Thus, of the six deaths in group DOX2 during the inductionperiod, three corresponded to males and three to females.Similarly, one death occurred in males and one in females inthe DAU3 group during the induction period, whilst for theDAU4 group the premature death occurred in a male rabbit.Cumulative mortality up to two weeks after completion ofthe induction period continued to increase in all groups(Figure 2). However, whilst one additional rabbit died inthe DOX2 group from 8 to 10 weeks, secondary to systemictoxicity, and 4more deaths occurred in theDAU3 group from10 to 12 weeks, 3 of these secondary to CHF, resulting in acumulative mortality of 58.3% and 100%, respectively, onlyone more death secondary to CHF occurred in the DAU4


Table 1: Selected biochemical parameters.

Parameter Basal Intermediate§ FinalCholesterol (mg/dL)

Control 41.5 ± 10.3 37.5 ± 10.2 44.7 ± 3.8DOX2 36.10 ± 11.25 70.46 ± 16.66bc#¶ 241.04 ± 59.5bc#¶

DAU3 40.80 ± 4.34 45.66 ± 6.40 79.73 ± 8.20bc

DAU4 35.24 ± 7.71 44.38 ± 4.05 59.08 ± 7.81bc

Triglycerides (mg/dL)Control 72.3 ± 22.9 45.3 ± 7.3 55.3 ± 7.1DOX2 68.23 ± 17.48 184.70 ± 47.74b¶ 405.88 ± 38.80bc¶

DAU3 78.09 ± 20.39 150.31 ± 65.32b¶ 446.22 ± 102.9bc¶

DAU4 72.50 ± 22.85 85.04 ± 12.35 115.88 ± 15.72bc

Creatinine (mg/dL)Control 0.86 ± 0.10 0.77 ± 0.11 0.83 ± 0.10DOX2 0.93 ± 0.06 1.08 ± 0.08 1.62 ± 0.22bc

DAU3 0.79 ± 0.04 1.06 ± 0.10 2.54 ± 0.50bc∗¶

DAU4 0.98 ± 0.10 0.92 ± 0.10 0.87 ± 0.07BUN (mg/dL)

Control 37.00 ± 4.95 38.54 ± 6.62 41.79 ± 8.50DOX2 36.43 ± 3.45 30.50 ± 3.74 39.40 ± 8.69DAU3 26.61 ± 2.99 30.92 ± 2.47 85.47 ± 12.34bc∗¶

DAU4 35.42 ± 4.57 28.35 ± 1.38 29.96 ± 2.50Total Proteins (g/dL)

Control 5.39 ± 0.21 5.37 ± 0.22 5.33 ± 0.24DOX2 5.48 ± 0.17 4.90 ± 0.37 3.92 ± 0.37bc¶

DAU3 5.31 ± 0.17 5.90 ± 0.30 4.42 ± 0.81bc

DAU4 5.55 ± 0.16 5.20 ± 0.12 4.96 ± 0.17AST (U/L)


DAU4 35.94 ± 5.73 23.30 ± 3.84 28.31 ± 5.67ALT (U/L)


DAU4 58.25 ± 13.44 45.03 ± 5.96 42.19 ± 6.88§Intermediate time point values were from blood samples obtained on week 8 of the study in control group, on week 6 in DOX2 group, on week 8 in DAU3group, and on week 4 in DAU4 group. BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Data expressed as mean ±SEM. Statistical significance is at p < 0.05; bcompared to basal value; ccompared to control; ∗compared to DOX2; #compared to DAU3; ¶compared to DAU4.

group from 6–8 weeks, resulting in a cumulative mortality of15.3%.

3.2. Incidence of DCM and CHF. None of the animals fromthe DOX2 group developed overt DCM or CHF, which onthe contrary were greatly affected by general toxicity. In theDAU3 group, 4 out of 6 animals (66%) developed overt DCMand CHF, of which two were males and two were females.In the DAU4 group, 12 out of 13 animals presented withovert DCM (92%), whilst CHF was confirmed in 8 animals(61%). In DAU4 group, the incidence of overt DCM and CHFaccording to sex of the animal revealed that 6 out of 7 malesand 6 out of 6 females presented with overt DCM, whilst HFwas present in 4 out of 7 males and 4 out of 6 females.

3.3. Biochemical Study. Selected biochemical parameters areshown in Table 1. No differences were observed between thedifferent groups at baseline. Triglycerides and cholesterol plustriglycerides were significantly increased at the intermediatetime point in theDOX2 andDAU3 groups, respectively, com-pared to basal values and control group, whilst in the DAU4group no significant differences were observed. Also, therewere slight increases in other biochemical parameters suchas creatinine for DOX2 and DAU3 groups at the intermediatetime point; however, these remained within the normal rangefor the species [24]. Groups DOX2 and DAU3 developedmarked changes in several biochemical parameters at thefinal time point of the study. Thus, rabbits in the DOX2 andDAU3 groups exhibited a significant increase in creatinine


and marked combined hyperlipidaemia associated with aconcomitant reduction of total proteins. In addition, theDAU3 group also showed significant increases in bloodurea nitrogen (BUN), aspartate aminotransferases (AST), andalanine aminotransferases (ALT). Apart frommild combinedhyperlipidaemia, no significant changes were observed inbiochemical parameters in the DAU4 group at the final timepoint (Table 1). Supplemental Table 1 in SupplementaryMate-rial available online at http://dx.doi.org/10.1155/2015/465342shows the subanalysis of selected biochemical parameters inmale and female animals from all groups at final time point.Consistent with the general analysis, both male and femalerabbits in the DOX2 and DAU3 groups had comparableincreases in creatinine values, marked combined hyperlipi-daemia, and reduction of total proteins in blood relative tocontrol group, suggesting that none of these alterations hadan underlying causality associated with sex of the animal.However, even though these changes were marked enoughcompared to control group, they were only significant inDOX2 group, provided the low 𝑛 of animals in DAU3 groupat this time point (type II error). Similarly, and in linewith thefindings of the general analysis, DAU3 group also presentedmarked alterations in BUN, AST, and ALT values in bothmales and females, although low 𝑛 at this time point in DAU3group leads to type II error (Supplemental Table 1). Male andfemale rabbits from the DAU4 group only exhibited mildhyperlipidaemia.

3.4. Haematological Study. In all AC treated groups, a sig-nificant reduction in red blood cells (RBC), haemoglobin,and haematocrit and increased red cell distribution width(RDW) were observed at the intermediate time point relativeto basal time point and control group. However, whilstin groups DOX2 and DAU4 these parameters returned tonormal levels at the end of the induction period, theseremained abnormal or even worsened in the DAU3 group atthe end of the induction period (Table 2). Consistent withincreased haematological toxicity with the DAU3 protocol,white blood cell (WBC) count also was significantly affectedat the intermediate time point compared to baseline valuesand those of control group, although these values returned tonearly normal at the end of the induction period (Table 2).

3.5. Echocardiographic Study. All groups of the study hadcomparable LVEF, FS, and total CSA at basal time point(Figures 3(a)–3(c)). A repeated measures test within thecontrol group demonstrated no significant differences onthese parameters of ventricular function at the different timepoints of the study. In the DOX2 group, a decrease in LVEFand FS was observed at the intermediate time point (6 weeks)and final time point (8 weeks), but this was only statisticallysignificant for LVEF at the intermediate time point (Figures3(a) and 3(b)). Whilst at the intermediate time point LVEF,FS, and total CSA remained unchanged in DAU3 and DAU4groups compared to basal time point and control group,all these parameters of ventricular function were markedlyand significantly reduced in rabbits from these groups atthe final time point (Figures 3(a)–3(c)). These changes werealso significantly different in DAU4 group when compared

to DOX2 at this time point (Figures 3(a)–3(c)). Subanalysisof ventricular function in all groups at the final time pointindicated thatAC-inducedmyocardial damage affectedmalesand females in DAU3 and DAU4 groups to a similar extent.Thus, marked alterations in LVEF, FS, and total CSA in malesand females were observed at final time point (SupplementalFigures 1(A)–1(C)). However, whilst these changes were sig-nificant in DAU4 group, low 𝑛 at final time point in DAU3group resulted in type II error. On the contrary, ventricularfunction remained equally unaffected in males and femalesfrom DOX2 group (Supplemental Figures 1(A)–1(C)).

3.6. Cardiac Troponin I Evaluation. At the basal time point,no differences in cTnI levels were observed among all groups.Significant elevations were observed at intermediate timepoints in all AC treated groups relative to basal time pointand control group (Figure 3(d)). The levels of cTnI furtherincreased at the final time point in all groups, although themost marked increase was observed in the DAU4 groupto the extent of being statistically significant at this timepoint compared to values for the DOX2 group (Figure 3(d)).The cTnI levels in males and females were also analysed atfinal time point in all groups (Supplemental Figure 1(D)).In line with the findings for the general population, markedelevations of cTnI were observed in both males and femalesfrom all groups, whilst males and females from DAU4 groupexhibited the highest levels of cTnI elevation relative tocontrol (Supplemental Figure 1(D)).

3.7. Histopathological Examination. Figure 4 shows repre-sentative photomicrographs of grades 1 and 4, within thespectrum of AC induced lesions in the myocardium, kidney,and liver, according to the grading scale used to determine thescore of lesions per organ [7]. The myocardial lesions fromall groups showed similar scores in grading scale as shownin Table 3. Thus, the myocardium of most animals presentedpredominantly with moderate myocytolysis associated withatrophy and degeneration of myofibrils and replacementfibrosis (Figures 4(b) and 4(c)). However, the extent ofmyocardial lesions was different for all groups. Whilst DOX2group animals presented mostly circumscribed lesions inisolated myofibrils, myocardial lesions in animals from theDAU3 group affected focal groups of myofibrils at 1 level,and animals of the DAU4 group exhibited lesions involvingmore extensive (diffuse) groups of myofibrils at 2 or morelevels (Table 3). Nonpurulent myocarditis associated with thepresence of mononuclear cell infiltration was also a frequentobservation in animals from all groups. Kidney and liverlesions from all groups showed similar scores, as well assimilar extension of lesions (see Table 4).

4. Discussion

An animal model of AICM suitable to test novel thera-pies aimed at ameliorating this condition not only shouldreproduce the cardiomyopathic effect of AC in scheduledintravenous injections, thus simulating clinical scenarios[25], but also should have low mortality at the end of


Table 2: Haematological parameters.

Parameter Basal Intermediate§ FinalRBC (106/𝜇L)

Control 5.95 ± 0.24 5.93 ± 0.12 6.12 ± 0.20DOX2 6.06 ± 0.20 4.76 ± 0.26bc 5.44 ± 0.74DAU3 6.29 ± 0.27 4.87 ± 0.19bc 3.83 ± 0.87bc∗¶

DAU4 6.33 ± 0.25 4.84 ± 0.20bc 5.23 ± 0.23Haematocrit (%)

Control 36.93 ± 1.76 36.88 ± 0.61 39.53 ± 0.90DOX2 37.11 ± 1.75 28.42 ± 1.54bc 33.29 ± 2.67DAU3 36.47 ± 1.10 29.56 ± 0.65 25.40 ± 3.56bc∗¶

DAU4 39.36 ± 0.98 29.16 ± 1.32b 33.94 ± 2.46Hemoglobin (g/dL)

Control 12.52 ± 0.47 11.75 ± 0.23 12.82 ± 0.30DOX2 12.21 ± 0.63 9.14 ± 0.32b 10.40 ± 1.23DAU3 12.18 ± 0.24 8.68 ± 0.24b 6.83 ± 1.08bc∗¶

DAU4 13.07 ± 0.44 9.19 ± 0.40b 9.57 ± 0.59bc

MCV (𝜇m3)Control 62.17 ± 2.12 62.35 ± 1.41 62.75 ± 1.78DOX2 61.18 ± 1.87 59.95 ± 3.18 62.40 ± 3.27DAU3 58.08 ± 1.52 60.88 ± 1.33 62.33 ± 5.03DAU4 62.29 ± 1.75 60.28 ± 1.36 64.73 ± 3.15

RDWControl 13.37 ± 0.74 13.95 ± 0.72 13.42 ± 1.14DOX2 13.08 ± 0.38 18.44 ± 1.15bc 17.32 ± 1.34bc

DAU3 13.48 ± 0.28 20.26 ± 0.83bc¶ 20.47 ± 1.44bc∗

DAU4 12.27 ± 0.41 15.98 ± 0.76bc 19.78 ± 0.78bc

WBC (103/𝜇L)Control 5.08 ± 1.22 4.86 ± 0.69 5.05 ± 0.82DOX2 5.77 ± 0.99 6.52 ± 1.14 5.53 ± 0.76DAU3 4.42 ± 0.64 2.88 ± 0.46bc∗¶ 7.41 ± 0.62bc∗

DAU4 5.43 ± 0.63 4.80 ± 0.72∗ 8.59 ± 1.92bc∗

Differential count (%)NeutrophilsControl 15.55 ± 2.52 17.57 ± 1.91 22.78 ± 5.20DOX2 18.20 ± 3.66 12.78 ± 2.49 25.70 ± 9.89DAU3 14.10 ± 2.19 10.18 ± 4.35 11.10 ± 0.95DAU4 31.16 ± 9.79 6.58 ± 1.60bc 27.44 ± 3.03

LymphocytesControl 68.55 ± 5.25 65.48 ± 2.97 63.50 ± 4.68DOX2 68.38 ± 4.55 70.63 ± 5.21 55.90 ± 10.34DAU3 73.27 ± 2.67 76.52 ± 5.47 72.20 ± 1.30DAU4 55.86 ± 8.73 82.56 ± 2.14 57.15 ± 3.47

§Intermediate time point values were from samples obtained on week 6 in DOX2 group, on week 8 in DAU3 group, and on week 4 in DAU4 group. RBC, redblood cells; MCV, mean corpuscular volume; RDW, red cell distribution width;WBC, white blood cells. Data expressed as mean ± SEM. Statistical significanceis at p < 0.05; bcompared to basal value; ccompared to control; ∗compared to DOX2; ¶compared to DAU4.

the induction period, thereby allowing sufficient time tocomplete experiments to evaluate their efficacy in recover-ing myocardial function and animal well-being. It also isdesirable that nonspecific toxicities, such as nephrotoxicity,which do not occur in humans treated with AC [18], arereduced or absent from the model to avoid the potential

confounding effect of comorbidities in the outcome of theexperiments. Such an animal model is currently unavailable.The present study presents an experimental protocol forAICM that generates a high percentage of animals with overtDCM and CHF with mild manifestations of systemic toxicityand very low mortality both premature (7.6%) and within


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BC∗

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Figure 3: Changes in echocardiographic parameters and cTnI values during induction of AICM. (a) Left ventricular ejection fraction (LVEF).(b) Fractional shortening (FS). (c) Total circumferential shortening area (CSA). (d) Cardiac troponin I (cTnI) levels. Data expressed as mean± SEM. Statistical significance is at 𝑝 < 0.05; (B), compared to basal; (C), compared to control; (∗), compared to DOX2; (#), compared toDAU3; (¶), compared to DAU4.

Table 3: Scores for grade and extension of histopathological lesionsin the heart.

Group Lesion grade Lesion extensionDOX2 2.65 ± 0.39 1.65 ± 0.44DAU3 2.63 ± 0.43 2.68 ± 0.47∗

DAU4 2.76 ± 0.32 3.18 ± 0.43∗#

Data expressed as mean ± SEM. Statistical significance is at p < 0.05;∗compared to DOX2; #compared to DAU3.

two weeks of completing the induction protocol (15.3%).Furthermore, the occurrence of CHF manifests insidiouslywhilst cardiogenic death did not occur abruptly at the endof the induction period, thus allowing time for functionalevaluation of the animal over an extended period of time.These qualities result in an improvement over previouslypublished protocols of AICM in rabbits particularly in thesetting of evaluation of novel therapies for AICM (e.g., stem

Table 4: Scores for grade and extension of histopathological lesionsin kidney and liver.

Group Lesion grade Lesion extensionKidney

DOX2 3.50 ± 0.71 3.50 ± 0.71DAU3 2.75 ± 0.50 3.50 ± 0.58DAU4 3.35 ± 0.57 3.17 ± 0.65

LiverDOX2 3.00 ± 0.10 3.00 ± 0.10DAU3 3.00 ± 0.75 3.00 ± 0.71DAU4 2.88 ± 0.90 3.04 ± 0.75Data expressed as mean ± SEM.

cell therapy). This refined model is in line with the principlesof the 3Rs and the guidelines of the National Centre forthe Replacement, Refinement and Reduction of Animals inResearch. Thus, lower premature mortality translates into


Haematoxilin and eosinGrade 1

Hea

rt

(a)


Hea

rt

(b)

Grade 4

Hea

rt

Masson’s blue trichrome

(c)


Kidn

ey

(d)

Grade 4Haematoxilin and eosin

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ey

(e)

Grade 4

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ey


(f)


Live

r

(g)

Grade 4Haematoxilin and eosin

Live

r

(h)

Grade 4

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(i)

Figure 4: Myocardial, renal, and hepatic lesions induced by anthracyclines. (a–c) Representative photomicrographs of myocardial damage.(a) DOX2 group, grade 1: primary damage of the left ventricular myocardium. Highly intense eosinophilia of the cytoplasm, pyknoticnuclei [P], and diffuse vacuolar degenerations in cardiomyocytes [∗] are present. (b) DAU3 group, grade 4: conspicuous disperse toxicmyocardial damage. Extensively vacuolated cardiomyocytes [∗] with intense eosinophilic cytoplasm are present. Necrotic cardiomyocytes[N] phagocytized by macrophages [M] were gradually replaced by proliferated connective tissue (e.g., thick wavy collagen fibers [arrows]).Mononuclear infiltrate [I] is present within the bundles of these fibers. (c) DAU3 group, grade 4: extensive fibrosis [arrows] in areas withmyocytolysis and necrosis of cardiomyocytes. (d–f) Representative photomicrographs of renal damage. (d) DOX2 group, grade 1: initial renaltubular necrosis. Lesions of nephrosis with tubular degeneration [∗] are present. (e) DAU3 group, grade 4: focal severe nephropathy. Lesionsof nephrosis with tubular degeneration [∗] and extensive necrosis [N] of tubular cells are frequent. Fibrosis [arrow] andmononuclear infiltrate[I] are present in these areas. (f) DAU3 group, grade 4: extensive fibrosis [arrows] in areas of toxic nephropathywith degenerations and tubularnecrosis. (g–i) Representative photomicrographs of hepatic damage. (g) DOX2 group, grade 1: initial damage of the liver. Some hepatocytesshow diffuse vacuoles inside cytoplasm [∗]. (h) DAU3 group, grade 4: hepatocytes revealed an extensive toxic damage. Necrotic hepatocytesare present [N] and abundant hepatocytes show numerous and extensive vacuoles inside cytoplasm [∗]. (i) DAU3 group, grade 4: diffusefibrosis [arrows] in areas with necrotic hepatocytes. (a-b), (d-e), and (f-g): haematoxylin and eosin; (c), (f), and (i): Masson’s blue trichrome.

lower number of animals required to complete a study(reduction), and reduced nonspecific toxicities translate intoreduced unnecessary suffering and improved well-being ofthe rabbits throughout the study (refinement).

One of the salient findings of our study is that, in contrastto the high attrition rate associated with the protocols of

induction used in DOX2 and DAU3 groups (Figure 2),the DAU4 group exhibited very low premature mortality(7.6%) during the induction period (with prematuremortalitydefined as death of the animal during the course of weekly I.V.administrations on each group). DAU4 group also exhibiteda significantly higher survival rate within two weeks after


completion of the induction protocol (Figure 2). Mortalityassociated with the induction of heart failure secondary toAICM in animal models using rabbits is rarely reported.Among the studies that do reportmortality using protocols ofinduction similar to those employed in our DOX2 and DAU3groups, the premature mortality is consistent with our find-ings (30–50%) [10, 14, 19]. The significantly lower mortalityin DAU4 group compared to that of DAU3 could be in partattributable to the fact that whilst higher weekly doses wereadministered in DAU4 (3mg/kg/week in DAU3 comparedto 4mg/kg/week in DAU4), these were administered over ashorter period of time (10 weeks in DAU3 versus 6 weeksin DAU4). This likely minimises the period of AC exposure,thus reducing the overall total cumulative dose at the end ofthe induction period (30mg/kg in DAU3 group compared to24mg/kg in DAU4). This resulted in comparable myocardialdamage between DAU4 group and DAU3 group, whilst itreduced extracardiac toxicity/compromise in DAU4 (see alsorelevant discussion below). Other protocols experimentallytrialled by us (e.g., 6mg/kg/week for 4-5 weeks) resulted inmortalities above 50%, primarily due to toxicity, and weretherefore stopped for ethical reasons and deemed inadequate.

Another important finding of our study is that theinduction protocol used in the DAU4 group is less proneto induce nonspecific toxicity such as nephrotoxicity andhepatotoxicity compared to the induction protocols used inthe DOX2 and DAU3 groups. Thus, whilst the DAU4 groupdid not exhibit changes in renal or hepatic function, animalsfrom the DOX2 and DAU3 groups presented changes inrenal function suggestive of nephrotic syndrome. Similarly,the DAU3 group showed increased levels of AST and ALT,suggesting compromised liver function in this group. Takinginto account these findings, it is likely that the high mortalityrate observed in the DOX2 and DAU3 groups is, at leastin part, explained by the increased incidence of nonspecifictoxic effects also observed in these groups (see also relevantdiscussion below). The sensitivity to the nephrotoxic effectsof AC in rodents, which is not observed in humans [18],has been extensively used as a model of nephrotic syndromewhich replicatesmost of the pathological features of focal andsegmental glomerulosclerosis seen in humans [26, 27]. Thenephrotoxicity observed in the DOX2 and DAU3 groups isconsistent with previous reports [10, 19, 28, 29]. Hepatotox-icity induced by doxorubicin in rabbits has been describedpreviously in one report [20]; however, we did not find alter-ations in biochemical markers of liver function in the DOX2group. Interestingly, previous studies that used a protocol ofinduction as in the DAU3 group in the present study didnot report alterations in liver function tests [10, 19]. Thereasons for these differences are unknown. Nevertheless, thechanges in AST and ALT at the final time point in the DAU3group suggest marked hepatocellular damage. Whilst thiscould be a consequence of direct AC-induced hepatotoxicity,other mechanisms could be partially responsible as well (seealso relevant discussion below). Given the shorter period ofexposure in the DAU4 group and the absence of nonspecifictoxicities/compromise such as nephrotoxicity and hepatotox-icity as suggested by elevations in biomarkers of kidney andliver damage, it is reasonable to conclude that these toxicities

are in part related to the length of exposure to AC, as opposedto cardiotoxicity, which is directly related to the cumulativedose received, although other potential explanations are alsopossible (see also relevant discussion below) [21].

In our study, haematological toxicity mostly affected redblood cells and was common to all groups of this studyeven though for most altered parameters this was transientsince this was observed at the intermediate time point andrecovered to normal or near normal levels at the final timepoint in the DOX2 and DAU4 groups, whilst it remainedaltered in the DAU3 group (Table 2). Haematological toxicityconsistent with myelosuppression (i.e., reduced number ofRBC, WBC, and platelets) has been reported previouslyin patients treated with AC [30, 31] and appears to be acommon feature of many antineoplastic drugs [32]. We didnot observe alterations in platelet count in any of the groupsstudied and only transient leukopenia in the DAU3 group,which is consistent with previous studies using protocols ofinduction similar to those used in the DAU3 group [10, 19].The finding that this is also observed in the DAU4 groupsuggests that this toxicity is independent of the scheduleddosing of AC. Indeed, given the high activity of AC inhaematological malignancies, this toxicity could be related totheir therapeutic benefits [1, 2].

Evaluation of left ventricular function by echocardio-graphy is a reliable noninvasive method for diagnosis ofAICM, which has good correlation with invasive catheter-based functional analysis in rabbit models of AICM [10]. Itis interesting to note that in this study the echocardiographicstudies were planned and supervised by European board-certified veterinary cardiologists with extensive clinical expe-rience, which adds value to the results and ensures therigorousness of the studies. Monitoring of LVEF and FSdemonstrated progressive reduction in these parameters onlyin the DAU3 and DAU4 groups. On the other hand, withthe exception of a transient decline in LVEF at the interme-diate time point (6 weeks) in animals of the DOX2 group,these parameters remained almost unchanged throughoutthe induction period, and none of the animals from thisgroup developed overt DCM or CHF. This is in contrast to aprevious study by Gava et al. who used an induction protocolas in theDOX2 group in our study, which found a progressivedecline in both LVEF and FS from as early as 6 weeks [15].Similar to our findings, another study, using even higherdoses of doxorubicin (doxorubicin 3mg/kg per week for 10weeks), also failed to demonstrate significant changes in leftventricular function [19]. The reasons for these discrepanciesare unknown at present, however, since our histopathologicalstudies indicate that this group exhibited the lowest score ofextension of myocardial damage (see also relevant discussionbelow); the cause of death in this groupwas exclusively due tosystemic toxicity, andmortality in the study of Gava et al. was70% (causes and timing of these deaths were not reported) asopposed to 50% in our study [15]; we suggest that this proto-col of induction may not be suitable for exploring the poten-tial benefits of novel therapies for HF secondary to AICM.

The incidence of overt DCM for the DAU3 and DAU4groups was 66% and 92%, respectively, which ultimatelytranslated to an incidence of CHF of 66% (group DAU3) and


61% (group DAU4). Of note, whilst the development of overtDCM and CHF in the DAU3 group was simultaneous in allcases, this was usually full blown severeCHF and very quickly(within a couple of days) resulted in the death of the animal, incontrast to a more insidious presentation in the DAU4 group,in which incidence of overt DCM was very high followed inmost cases within days by CHF, which persisted for severalweeks before death ensued. Other parameters of myocardialdamage were also more exacerbated in DAU4 group. Thus,whilst an elevation of cTnI, a marker of myocardial cellulardamage, was observed in all groups, much higher levelswere detected in the DAU4 group at the final time point(Figure 3(d)). Of note, none of the differences observed inLVEF, FS, total CSA, or cTnI between DAU3 and DAU4groups were statistically significant, despite a clear trend(Figure 3). Elevation of cTnI has been documented previouslyin rabbits treated with an induction protocol as in the DAU3group in our study [33].

Histopathological examination revealed that whilst ani-mals of all groups have a similar grade of myocardial lesions,these were more conspicuous in the DAU4 group, since thescore of extension of lesions observed in this group was sig-nificantly higher than that in the other groups (Table 3). Thehistopathological findings of the heart, kidney, and liver in theDOX2 andDAU3 groups are consistent with previous reports[10, 15, 19]. Of note, despite marked elevations in surrogatebiomarkers of renal injury in DOX2 and DAU3 groups andof liver injury in DAU3 group compared to DAU4 groupand control group at final time point, the scores of kidneyand liver lesions in grade and extension of damage amongstthe AC treated groups were similar upon histopathologicalexamination (Table 4). Whilst nephrotoxicity and hepatotox-icity have been described in rabbits treated with AC, thesemay not completely explain this lack of correlation betweenblood biomarkers and histopathology findings and it is worthelucubrating about other potential explanations. The rabbitsfrom DOX2 group were greatly affected by general toxicityand exhibited the highest mortality (50%). Rabbits fromthis cohort were often observed to be asthenic, subsequentlydeveloped anorexia, and near the end of the protocol (or endof their life) appeared cachectic, which likely resulted from anincreased catabolic state, leading to emaciation, a conditionoften associated with multisystem organ failure, includingkidney damage. These observations could explain, at least inpart, the elevated creatinine observed in this group. On theother hand, 66%of rabbits fromDAU3group developed overtDCM which was quickly followed by progression to a severeform of CHF, which resembled acute severe decompensatedCHF, with death ensuing within days. Hepatic (retrograde)congestion could in part explain the increased elevationsof ALT and AST. Hypoperfusion secondary to poor pumpperformance and neuroendocrine activation (which is moremarked in some forms of CHF such as acute decompen-sated CHF) and associated peripheral and renal-splanchnicvasoconstriction could contribute to increased renal damage(even though renal damage secondary to CHF (cardiorenalsyndrome) is multifactorial) [34] and thus explain to someextent elevated biomarkers of kidney injury (e.g., creatinineand BUN) in the setting of AC treatment in this group.

Some long-term association studies indicate that amongchildhood cancer survivors treated with AC girls appearto have increased risk of developing long-term AICM [35,36]. Interestingly, amongst adults, men appear to be moresensitive than women to AICM [37]. Gender differences alsoappear to play a role in rodent models of AICM and ACnephropathy, with males and ovariectomized females beingmore affected than females [38–40]. Although evidence is stillinconclusive, with recentmetaregression analyses and associ-ation studies indicating that female sex is not a risk factor forAICM [41, 42], most of the current available evidence pointsto the conclusion that oestrogen could have a protective rolein AICM. We did not find any clear difference attributableto gender in terms of mortality, incidence of overt DCM, orcongestive heart failure, as suggested by a similar percentageof males and females from DAU3 and DAU4 groups beingaffected in the present study. Similarly, at the final time point,we did not find any significant differences in biomarkers ofcardiac (i.e., LVEF, FS, total CSA, and cTnI levels), renal (e.g.,creatinine and BUN), or hepatic (i.e., AST and ALT) functionamongst males and females on each of the AC treated groups(Supplemental Table 1 and Supplemental Figure 1). Thus, incontrast to experimental studies in rodents, taken together,our data do not support a link between gender differencesand AC toxic sensitivity in rabbits. Of note, beyond a clearACmediated toxicity, our studywas not powered to assess sexdifferences to AC sensitivity in theDAU3 group, thus limitingour conclusions in this respect.

Taken together, our results suggest that during theinduction of AICM the overall extracardiac physiology ofrabbits from DOX2 and DAU3 groups was more severelycompromised compared to that of the rabbits from DAU4group, whilst DAU4 induced comparablemyocardial damageto that of DAU3 group (Figure 3). We believe that therefined induction protocol used in the DAU4 group could bebetter suited for evaluation of novel therapies (e.g., stem celltherapy) aimed at ameliorating the condition, given the highincidence of overt DCM and the insidious and more stableform of CHF that follows, which translates into increasedsurvival at the end of the induction period. As a note addedin proof, we have recently reported that, using the protocolof induction as in DAU4 group and successful induction ofAICM, stem cell therapy with amniotic membrane-derivedmesenchymal stem cells (AM-MSC), administered percuta-neously via intramyocardial injection, significantly improvedventricular function at 2 and 4 weeks after transplant andalso significantly improved survival compared with controlgroup [43]. We also believe that the protocol used in DAU3group is still very valuable in evaluation of novel cardiopro-tective drugs, aimed at preventing development AICM in thepreclinical setting and serving as a model for the study ofpathophysiological aspects of AICM.

5. Conclusion

Our results indicate that, compared to other protocols ofinduction of AICM (as in the DOX2 and DAU3 groups), aprotocol using daunorubicin 4mg/kg per week for 6 weeks(as in the DAU4 group) results in a high percentage of


animals with overt DCM (92%) and CHF (61%), with mildmanifestations of nonspecific systemic compromise, very lowpremature mortality (7.6%) during the induction period,and low cumulative mortality within the two weeks aftercompletion of the induction protocol, which translated intoa significantly higher survival rate at this stage, whilst devel-opment of CHF was more insidious (compared to the DAU3group, in which abrupt development of CHF was quicklyfollowed by death of the animals). We propose that thisrefinement of the model of AICM in rabbits, which translatesinto a more predictable cardiotoxicity, represents a usefultool for the preclinical evaluation of novel therapies (e.g.,drugs or stem cell therapy) for the treatment of heart failuresecondary to AICM, as well as the study of the molecularmechanisms involved in the development of AICM. Thisrefinement is also in line with the guidelines of the NationalCentre for the Replacement, Refinement and Reduction ofAnimals in Research and the 3Rs principles, since the reducednonspecific toxicity and lower mortality would ultimatelytranslate into improved overall well-being of the animalsand reduced amount of animals required for this type ofresearch. However, we believe that the protocol used inDAU3 group is still very valuable in evaluation of novelcardioprotective drugs, aimed at preventing developmentAICM in the preclinical setting, and for the study of thepathophysiology of AICM.

Abbreviations

AC: AnthracyclinesAICM: Anthracycline-induced cardiomyopathyCHF: Congestive heart failureDCM: Dilated cardiomyopathyLVEF: Left ventricular ejection fractionFS: Fractional shortening.

Disclosure

The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the paper.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Authors’ Contribution

Conception and design of study and this paper were byJesus Talavera, Alejandro Giraldo, and Jose M. Moraleda.Conducting experiments and participating in data collec-tion were by Jesus Talavera, Alejandro Giraldo, and MarıaJosefa Fernandez-Del Palacio. Histopathological study wasby Obdulio Garcıa-Nicolas and Juan Seva. Data analyseswere performed by Jesus Talavera and Alejandro Giraldo.Drafting of the paper was performed by Alejandro Giraldo,Jesus Talavera, and Gavin Brooks. Revision of the paper wasperformed by all authors. All authors gave final approval to

the paper.The first two authors (Jesus Talavera and AlejandroGiraldo) contributed equally to this work.

Acknowledgments

Theauthors thankGiorgia Santarelli for the excellent researchsupport provided during the collection of data.This studywassupported by Fundacion Seneca, Agencia de Ciencia y Tec-nologıa, Region de Murcia, Spain (Jesus Talavera) (Grant no.11935/PI/09), Red de Terapia Celular, ISCIII-Sub. Gral. Redes,VI PN de I+D+I 2008-2011 (Grant no. RD12/0019/0001) (JoseM. Moraleda), Cofinanciado con Fondos Estructurales de laUnion Europea (FEDER) (Jose M.Moraleda), and Universityof Reading, United Kingdom (Alejandro Giraldo and GavinBrooks) (central funding).

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

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Research and TreatmentAIDS


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Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

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